Quantitative assay of the carbohydrate in urokinase-type plasminogen activator by lectinenzyme immunoassay

Abstract

We developed an enzyme immunoassay (EIA) for the measurement of Asn302-linked carbohydrate in urokinase-type plasminogen activator (u-PA) using peroxidase (HRP)-labelled lectins. u-PA antigen in the sample was immunologically bound to microtitre plate wells by anti-u-PA IgG and the binding of HRP-labelled lectins [Con A (Concanavalin A), WGA (wheat germ agglutinin), PNA (peanut agglutinin), CSA (Scotch broom), GS-I (Groffonia simplicifollia) and SBA (soybean agglutinin)] to the carbohydrate of u-PA was measured. The lectin-EIA was dose-dependent in the range 6-6000 IU/ml of u-PA using Con A and WGA. The assay did not detect the carbohydrate of bovine albumin, ovalbumin, human albumin, plasminogen, tau-globulin and fibrinogen. The binding of HRP-labelled Con A and WGA to the carbohydrate of u-PA was specifically inhibited by alpha-methylmannose and N-acetylglucosamine respectively. Endo F treatment of the carbohydrate of u-PA decreased the binding of Con A and WGA. The carbohydrate of u-PA obtained from chest fluid, ascites and U937 cell culture medium reacted with Con A and WGA by this assay forming a band in the 55 kDa region. These results suggest that the lectin-EIA method is suitable for the assay of the carbohydrate in the B-chain of u-PA.